SAM 512.06

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 512

Supplemental Assay Method for the Determination of Formaldehyde in 

Veterinary Biologics (Ferric Chloride Test)

Date: April 5, 2022

Number: SAM 512

Supersedes: SAM 512.05, February 8, 2016

Standard Requirement: 9 CFR 113.100 (f), 9 CFR 113.200 (f) 

Contact: Email: Methodsrequest.notification@usda.gov

Phone: Center for Veterinary Biologics, 515-337-6100

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product 

by USDA and does not imply its approval to the exclusion of other products that may be suitable.
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Supplemental Assay Method for the Determination of Formaldehyde in Veterinary Biologics 

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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of Standards

3.3 Preparation of vaccines containing oil emulsion

4. Performance of the Test

5. Calculations and Interpretation

6. Report of Test Results

7. References

8. Summary of Revisions
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Supplemental Assay Method for the Determination of Formaldehyde in Veterinary Biologics 

(Ferric Chloride Test)

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1. Introduction

This Supplemental Assay Method (SAM) describes how total formaldehyde is determined based 

on the reaction of formaldehyde with Methylbenzothiazolone hydrazone hydrochloride (MBTH). 

The method involves: a) the combination of MBTH and formaldehyde to give one product; b) 

the oxidation of excess MBTH to give another product; and c) the combination of these two to 

give a blue chromophore which is measured at 628 nm.

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 Spectrophotometer with appropriate accessories, i.e., cuvettes

2.1.2 Common laboratory apparatus and glassware – pipettes, pipettors with 

tips, screw cap tubes, Class A volumetric flasks, linear graph paper

2.2 Reagents

Equivalent reagents or supplies may be substituted for any brand name listed below.

2.2.1 Ferric chloride-sulphamic acid reagent. A solution containing 10 g/L of 

ferric chloride and 16 g/L of sulphamic acid.

2.2.2 Methylbenzothiazolone hydrazone hydrochloride reagent. (MW 233.7). 

(CAS 149022-15-1). 3-Methylbenzothiazol-2(3H) one hydrazone hydrochloride 

monohydrate. An almost white or yellowish crystalline powder. mp: about 270°C. 

A solution containing 0.5 g/L.

WARNING: This solution is not stable and should be prepared fresh prior to 

testing.

2.2.3 Suitability for determination of aldehydes. To 2 mL of aldehyde-free 

methanol, add 60 μL of a 1 g/L solution of propionaldehyde in aldehyde-free 

methanol and 5 mL of a 4 g/L solution of Methylbenzothiazolone hydrazone 

hydrochloride. Mix; allow to stand for 30 minutes. Prepare a blank omitting the 

propionaldehyde solution. Add 25.0 mL of a 2 g/L solution of ferric chloride to 

the test solution and to the blank, dilute to 100.0 mL with acetone R and mix. 

Measure absorbance of the test solution on a spectrophotometer at 660 nm in a 
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Supplemental Assay Method for the Determination of Formaldehyde in Veterinary Biologics 

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1-cm cell using the blank as compensation liquid. The absorbance of the test 

solution must be greater than or equal to 0.62 absorbance units.

2.2.4 Formaldehyde solution, containing not less than 34.5 percent w/v and not 

more than 38.0 percent w/v of formaldehyde (CH2O)

2.2.5 Isopropyl myristate, analytical grade

2.2.6 Hydrochloric acid (1 M), analytical grade

2.2.7 Chloroform, analytical grade

2.2.8 Sodium chloride (9 g/L and 100 g/L aqueous solutions), analytical grade

2.2.9 Polysorbate 20, analytical grade

3. Preparation for the Test

3.1 Personnel qualifications/training

Technical personnel must have experience or training in this protocol. This includes 

working knowledge of the use of general laboratory equipment and glassware; and 

specific training in the operation of the laboratory equipment listed in Section 2. 

3.2 Preparation of Standards

Prepare formaldehyde standards of 0.25, 0.50, 1.00 and 2.00 g/L by diluting 

formaldehyde solution (Section 2.2.4) with water in suitable volumetric flasks.

3.3 Preparation of vaccines containing oil emulsion

If vaccine to be examined is an oil emulsion, the emulsion should be broken by a suitable 

method. The formaldehyde concentration in the aqueous phase should be measured. The 

following separation techniques have been shown to be appropriate.

3.3.1 Add 1.00 mL of vaccine to 1.0 mL of isopropyl myristate and mix. To the 

mixture, add 1.3 mL of 1 M hydrochloric acid, 2.0 mL of chloroform, and 2.7 mL

of 9 g/L sodium chloride. Mix thoroughly. Centrifuge at 15,000 g for 60 minutes. 

Transfer the aqueous phase to a 10-mL volumetric flask and dilute to volume with 

water. Use the diluted aqueous phase for the test for formaldehyde. If this 

procedure described fails to separate the aqueous phase, add 100 g/L of 

polysorbate 20 to the sodium chloride solution and repeat the procedure, but 

centrifuge at 22,500 g.
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Supplemental Assay Method for the Determination of Formaldehyde in Veterinary Biologics 

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3.3.2 Add 1.00 mL of vaccine to 1.0 mL of a 100 g/L solution of sodium 

chloride and mix. Centrifuge at 1000 g for 15 minutes. Transfer the aqueous 

phase to a 10-mL volumetric flask and dilute to volume with water. Use the 

diluted aqueous phase for the test for formaldehyde.

3.3.3 Add 1.00 mL of vaccine to 2.0 mL of a 100 g/L solution of sodium 

chloride and 3.0 mL of chloroform and mix. Centrifuge at 1000 g for 5 minutes.

Transfer the aqueous phase to a 10-mL volumetric flask and dilute to volume with 

water. Use the diluted aqueous phase for the test for formaldehyde.

Note: Volumes used for breaking emulsions are for the purpose of 

illustration. Volumes may differ subject to proportional adjustment of the 

volumes of other reagents used in the extraction process.

4. Performance of the Test

4.1 To 0.50 mL of a 1 in 200 dilution of the vaccine to be examined (if emulsion, use 

0.50 mL of a 1 in 20 dilution of the diluted aqueous phase), and to 0.50 mL of 1 in 200 

dilution of each of the formaldehyde standards, add 5.0 mL of the Methylbenzothiazolone 

hydrazone hydrochloride reagent. Close the tubes, shake, and allow to stand for 60 

minutes.

4.2 Add 1 mL of ferric chloride-sulphamic acid reagent and allow to stand for 15 

minutes.

4.3 Measure absorbance of vaccines and standards on a spectrophotometer at the 

maximum at 628 nm in a 1-cm cell, using the reagent blank as compensation liquid.

5. Calculations and Interpretation

Use linear regression to fit the standard curve; a valid standard curve has a coefficient of 

determination (R2) equal to or greater than 0.97. If the standard curve is valid, calculate total 

formaldehyde concentration (g/L) by interpolation from the standard curve.

6. Report of Test Results

Test results are reported following the current standard operating procedures.
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7. References

7.1 Title 9, Code of Federal Regulations, parts 113.100(f) and 113.200(f), U.S. 

Government Printing Office, Washington, DC.

7.2 Testing of Residual Formaldehyde, VICH GL25 (Biologicals: Formaldehyde), 

Final, April 2002.

8. Summary of Revisions

Version .06

• Coversheet updated.

• 5. Revised to correct terminology.

Version .05

• Updated Contact information.

Version .04

• The Contact information has been updated.

• 3.1: Personnel qualifications/training has been updated.

• 7: The Reference of “Testing of Residual Formaldehyde” has been added.

• The entire document has been revised to reflect current practices.

Version .03

• The document number has been changed from TCSAM0512 to SAM 512.

Version .02

This document was revised to clarify the practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the name of the contact person has changed.